PERIODONTAL PATHOGENESIS

Presented by:-
Richa
Sharma
 INTRODUCTION

Pathogenesis is defined as the origination and development of a

disease. It is a step-by-step process that leads to the
development of a disease, resulting in a series of changes in
the structure and function. It is the process by which the
etiologic factor causes the disease.
Carranza 11th edition
 HISTOPATHOLOGY OF PERIODONTAL
DISEASE Periodontal pathogenesis
histologic appearance

Clinically healthy Inflamed periodontal and

gingival tissue
gingival tissue

Even in the gingival that appear clinically normal there is a chronic low-grade
challenge presented by sub-gingival plaque bacteria.

Essential mechanism to combat the microbial challenge to

prevent bacteria and their products from infiltrating tissues.

But an excessive, dysregulated, immune-inflammatory response for a

given
bacterial challenge leading to increased tissue breakdown.
 CLINICALLY HEALTHY GINGIVAL TISSUES
Clinically healthy gingival tissue is not inflamed, pink in appearance, not
swollen and firmly attached to the underlying tooth/bone, with minimal
bleeding on probing. The Dento-gingival junction is a unique anatomic
feature whose function is the attachment of the gingiva to the tooth.
DENTO-GINGIVAL
JUNCTION

Epithelial portion Connective tissue portion

Both are of fundamental importance in periodontal
pathogenesis.
Gingival epithelium
EPITHELIAL Junctional epithelium
Sulcular epithelium
STRUCTURE
GINGIVAL
EPITHELIUM
 Stratified squamous keratinized epithelium
 Continuous with the sulcular epithelium at the gingival crest/
gingival margin.
 Covers the gingiva and forms the clinically visible gingival tissues.
 Covers both the free and attached gingival tissues.

SULCULAR EPITHELIUM
 Stratified squamous epithelium.
 Non-keratinized
 Faces the tooth but it not attached to it.
 Forms the soft tissue lining of the gingival sulcus or periodontal
pocket.
 JUNCTIONAL EPITHELIUM
Unique epithelial structure
Specialized for the purpose of attachment to the tooth.
Unlike other epithelial tissues elsewhere in the body, there is no
opportunity for sloughing of cells from the surface.

The extracellular spaces between the junctional epithelium are also

greater than other epithelial tissues, with intercellular comprising
approximately 18 % of the volume of the epithelium.
This is a result of a lower density of desmosomes in the junctional
epithelium compared to the gingival epithelium, and the hence
junctional epithelium is “intrinsically leaky”.

This has great relevance in periodontal pathogenesis, as the

widened permit migration of neutrophils and macrophages from the
gingival connective tissue to enter sulcus to phagocytose bacteria,
as well as ingress of bacterial products as well as antigens.
 According PAGE and SHROEDER in 1976

Low grade inflammation Sub-gingival

micro flora

Overt clinical signs of gingivitis do not develop because innate and

structural defense mechanisms, including the following:-
 The maintainence of an intact epithelial barrier.
Outflow of GCF from the sulcus.
Sloughing of surface epithelial cells of the junctional and sulcular
epithelium.
Presence of neutrophils and macrophages in the
sulcus, phagocytosing bacteria.
Antibodies in the GCF.
If the plaque accumulation increase so that these defense mechanism are
overwhelmed, then inflammation and the classic clinical signs of
gingivitis develop.
 HISTOPATHOLOGY OF GINGIVITIS AND PERIODONTITIS
Infiltration of the tissues by numerous defense cells, particularlyneutrophils,
macrophages, plasma cells, and lymphocytes.

Accumulation of defense cells and the extracellular release of their

destructive enzymes

There is disruption of the normal anatomy of the connective tissues

resulting in collagen depletion and subsequent proliferation of junctional
epithelium.

Vasodilatation and increased vascular permeability leads to increased leakage

of fluid out of the vessels, and

facilitates the passage of defense cells from the vasculature into the tissues
which appear erythematous and edematous.
 HISTOLOGIC STAGES OF GINGIVITIS AND PERIODONTITIS
Page and Schroeder; Lab Invest,
INITIAL LESION 1976 EARLY
LESION
gingivitis that is evident
 Develops within 2 to 4 days
clinically
No inflammation is evident
Develops after about 1 or 4
microscopically
to 7 days week of continued
This lesion corresponds to
plaque accumulation
histologic picture of clinically
Increased vascular
healthy gingival tissues.
permeability, vasodilatation ,
 Slightly elevated vascular
GCF flow.
permeability
 Large no. of
and vasodilatation.
leukocytes
infiltratingmainly neutrophils
GCF flows out of the sulcus
& lymphocytes.
leading to flushing action.
 Degeneration of fibroblasts.
Migration of leukocytes,
 Collagen
primarily neutrophils in small
resulting
destruction,
in collagen depleted
numbers through the gingival
areas of the connective tissue.
connective tissues into the
Proliferation of junctional
sulcus.
and sulcular epithelium
into
 Established Lesion Advanced Lesion

It marks the transition from

This lesion develops from gingivitis to periodontitis.
14 to 21 days add corresponds  This transition is determined
to chronic gingivitis. by bacterial challenge, host
Dense inflammatory cell inflammatory and
infiltrate i.e. plasma bacterial
response challenge
lymphocytes, neutrophils cells, susceptibility factors. and
Elevated release of MMPs Predominance inflammatory
and lysosomal contents from cells in the connective
neutrophils. tissues.
Significant Predominant immune
collagen depletion and cells are plasma cells.
proliferation of epithelium. The basal lamina
 Formation of pocket differs
oppose fromconnective
other basal
tissue in
epithelium containing large that
laminae type
that IV is
numbers of neutrophils. absent.
collagen
 INFLAMMATORY RESPONSES IN THE PERIODONTIUM
During the inflammatory response there are specific molecules which signal
tissue damage as the inflammatory response develops:-

Sub-gingival micro flora host immune-inflammatory response

The bacteria are important because they drive and perpetuate the
inflammation but they are responsible for a relatively small proportion of
tissue damage.
CARRANZA 11thedition

1) Microbial Virulence Factors

2) Host-Derived Inflammatory Mediators
3) Role of Specific Inflammatory Mediators
 MICROBIAL VIRULENCE FACTORS
1) LIPPOLYSACCHARIDE

Lipid component Polysaccharide component

(Lipid A)

 Found in the outer membrane of gram-negative bacteria

 Act as endotoxins

 Elicit a strong immune response

 Have an important role in maintaining the structural integrity of bacterial

cells.
Immune system recognizes LPS through toll-like receptors(TLRs) .
TLRs are cell surface receptors that recognize microbe-associated molecular
patterns (MAMPs) which are conserved molecular structure located in
diverse pathogens.
TLR-4 recognizes LPS from gram-negative bacteria and functions as part of a complex of
cell surface molecule, including CD14 and MD-2 ( known as lymphocyte antigen 96).
Like LPS component of gram-positive cell walls lipoteichoic acid (LTA) also stimulates
immune response although less potently than LPS.

CD14/TLR-4/MD-2 LPS or LTA

increased production of inflammatory mediators

increased vasodilatation and vascular permeability, recruitment of

inflammatory cells by chemotaxis,

release of pro-inflammatory mediators by leukocytes which recruite to the area of

inflammation.

LPS in particular is a key importance in initiating and sustaining inflammatory

responses in
gingival and periodontal tissues.
2) Bacterial Enzymes and Noxious Products

Plaque bacteria produce a number of metabolic waste products which

contribute directly tissue damage. These noxious agents include ammonia,
hydrogen sulfide and short chain fatty acids.

These substances have pro-found effect on host cells which are as follows:-
a) The short chain fatty acid aid P.gingivalis infection through tissue
destruction and also create a nutrient supply for the organism the
bleeding into periodontal pocket.
b) Plaque bacteria produces Proteases which are capable of break downing
the structural components of the periodontium such as collagen, elastin.
These digest proteins and provide nutrition to bacteria.
c) produces 2 classes of cysteine proteases known
P.gingivalis as GINGIPAINS

Lysine-specific gingipain Arginine-specific gingipain

Rgp A Rgp B

The gingipains modulate the immune system and disrupt

immune-
inflammatory responses. Potentially leading to increased tissue damage.

These can reduce the concentration of cytokines and digest and inactivate
TNF-α.

Stimulate cytokine secretion via activation of protease activated receptors

(PARs) thereby stimulating cytokine secretion.
3) Microbial Invasion
 Periodontal pathogens such as P. gingivalis and a.a. invade the gingival

tissues including connective tissue. A. Actinomycetemcomitans can invade

epithelial cells and persist intracellularly.
J Periodontol 67:291-297,1996
 Acc. to some investigators tissue invasion is a passive translocation process

and according to the others it is an active process. But the presence of

bacteria justifies the use of antibiotics to some extent which are protected
from mechanical disruption by root surface debridement.

 The bacteria in the tissues represent the reservoir of re-infection after non-
surgical management. However the clinical relevance of bacteria being
present in the tissues is better defined, it is inappropriate to make clinical
treatment decisions (e.g., whether to use adjunctive systemic antibiotics) on
this premise alone.
4) FIMBRIAE
The fimbriae of certain bacteria particularly P. gingivalis play important role in
pathogenesis.
Major fimbrial structural component of P.gingivalis, Fim a

Secretion of IL-6. IL-8 and

TNF-α
stimulation of nuclear factor
NF-κB and IL-8 in gingival
epithelial cell line Interaction with complement
receptor-3 to activate
intracellular signalling
pathways that inhibit IL-12
production.
This may be of clinical relevance as IL-12 is important in activating natural killer
(NK) and CD8+ cytotoxic T-cells which themselves are important in killing
P.gingivalis – infected host cells such as epithelial cells.
5) Bacterial Deoxyribonucleic Acid and Extracellular Deoxyribonucleic
Acid
Bacterial DNA stimulates the immune cells via TLR-9 which recognize s
hypomethylated CpG regions of DNA. These are regions of DNA at which a
cytosine nucleotide is found next to a guanine nucleotide separated by phosphate
molecule.
Extracellular DNA plays important role in the development and structure of the
biofilm formed by oral bacteria and there are majority of eDNA released after cell
lysis.

This donated DNA is of prime importance as it acts as a means of increasing genetic

diversity , if taken up by other bacteria.

Contributing to antigenic variation and spread of antibiotic resistance,

Modulation of host immune response.

Hence eDNA is a source of genetic information for

naturally transformable
bacteria in biofilm and also acts as a stimulus for host
immunity.
 HOST DERIVED INFLAMMATORY MEDIATORS

BY-STANDER DAMAGE
The inflammatory and immune processes that develop in the periodontal
tissues in response to the long-term presence of sub-gingival bio-film are
protective by intent but result in considerable tissue damage.

Majority of the tissue damage in periodontitis derives from the excessive and
dysregulated production of a a variety of inflammatory mediators and
enzymes which are broadly classified as follows:-

 CYTOKINES
 PROSTAGLNDINS
 MATRIXMETALLOPROTEINASES
 CYTOKINES
Cytokines play a fundamental role in inflammation and are key inflammatory
mediators in periodontal disease.
Cytokines are soluble proteins, secreted by cells, which act as messenger
molecules that transmit signals to other cells and initiate intracellular
signalling cascades resulting in phenotypic changes in cell.
Cytokines are produced by- neutrophils, macrophages & lymphocytes
alongwith resident cells including fibroblasts and epithelial cells.

CYTOKINES

Profound biologic effects Significant overlapping activity

 signal, broadcast and amplify
immune response
 Mediate connective tissue
and
alveolar bone destruction through
induction of fibroblast & osteoclasts
production of proteolytic enzyme.
Cytokines function in complex
networks that involve both
pro-inflammatory and anti-
inflammatory effects that brings
together both the aspects of
Innate and acquired immunity.
IL-1 Family Cytokines
IL-1β

Key role in Innate immune Secretion of

inflammation response mediators

IL-1β stimulates the synthesis of PG2, PAF, NO that causes vascular

changes associated with inflammation.
It increases the expression of ICAM-1 on endothelial cells
and stimulate the secretion of chemokines.
It synergises with other proinflammatory cytokines and PGE2 to
induce bone resorption.
It has a role in adaptive immunity and regulates the development of
antigen-presenting cells such as dendritic cells.
b) IL-1α intracellular protein & potent bone resorbing
factor

An “alarmin’’ to signal the immune system during cell damage.

c) IL-1Ra - binds to IL-1 receptor (IL-R1).
 However, binding of IL-Ra does not result in
signal transduction, therefore IL-1Ra antagonizes the action of
IL-1β.
 It is important in regulating inflammatory and considered
to be an anti-inflammatory cytokine.
d) IL-18 - interacts with IL-1β. It is produced by
stimulated macrophages and monocytes.
It results in pro-inflammatory responses including activation of
neutrophils.
It is a chemoattractant for T-cells, and it interacts with IL-12 and IL-
15 to induce Th1 cells cell-mediated immunity.
e) TNF-α: it is secreted by activated macrophages, particularly in
response to bacterial LPS.

Proinflammatory effects:
• Stimulation of endothelial cells to express selectins
that facilitate leukocyte recruitment.
• Activation of IL-1b production.

• Induction of PGE2 by macrophages and fibroblasts.

• GCF levels of TNF-α increases as gingival

inflammation develops.
 ANTI-INFLAMMATORY CYTOKINES
 Anti-inflammatory cytokines include IL-10, TGF-β, IL-1Ra,
IL-1F5, IL-1F10.
 IL-10 family of cytokines have multiple pleiotropic effect and
possess immunosuppressive properties.
 It is produced by T reg cells, monocytes, and B-cells and it
suppresses the secretion of Th1 cells, Th2 cells, monocytes,
macrophages.
 TGF-β is a growth factor and functions as a cytokine with
immunoregulatory role.
 It plays important role in repair and regeneration, apoptosis
and angiogenesis.
 CHEMOKINES
These are cytokine-like molecules that are characterized by their
chemotactic activity.
Chemokines orchestrate leukocyte recruitment in physiologic
and pathologic conditions and are therefore important in
periodontal pathogenesis.
The chemokine CXCL8, also known as IL-8, is found in the
areas of plaque accumulation as well as GCF.
Interaction between bacteria and cytokines leads to up-
regulation of IL-8 which is responsible for development of
chemotactic gradient and also stimulates neutrophil migration
in the sulcus. Similar chemotactic gradient is present in
periodontally healthy tissues.
2) Prostaglandins - It is a group of lipid compounds derived
from arachidonic acid, mainly mediated by cyclo-oxygenase
and lipo-oxygenase pathway..
 The cells primarily responsible for PGE2 production in the
periodontium are macrophages and fibroblasts.
 Its level is increased in the tissues and in GCF at sites
undergoing periodontal attachment loss.
 These are important mediators of inflammation,
particularly, PGE2, which results in vasodialation and
induce cytokine production.
 COX-2 is upregulated by IL-1b, TNF-a and LPS leading to
increased production of PGE2 in inflammed tissue.
 PGE2 results in induction of MMP’s and osteoclastic bone
resorption and has a major role in contributing to tissue
destruction that characterizes periodontitis.
3) Matrix metalloproteinase's- It is a family of proteolytic enzymes
that degrade extracellular matrix molecules such as collagen,gelatin
and elastin.

It is produced by variety of cells including macrophages, neutrophils,

fibroblasts, epithelial cells, osteoclasts.

Latent form Activated form

Proteases such as Cathepsin G
produced by neutrophils.

These MMP’s are inhibited by

• Tissue inhibitor of metalloproteinase

(TIMPs)
• Glycoproteins α-1 antitrypsin and α-2

macroglobulin.
 Inflamed periodontal tissues

excessive quantities of MMPs

large quantities of infiltrating cells

MMPsInhibitors

Development of collagen depleted areas within

connective tissue
The predominant MMPs in periodontitis, MMP-8 and MMP-9
are secreted by neutrophils and are very effective in degrading
type I collagen, the most abundant type in the PDL.
 If inflammation becomes more extensive because
of increase in bacterial challenge

Increased vascular permeability, vasodilatation leading to

edema, erythema.
 Gingival swelling, slight deepening of sulcus
further compromises plaque removal.

Increased infiltration of inflammatory cells leads to

development of collagen depleted areas below epithelium
and the epithelium proliferates to maintain its integrity.
 If the bacterial challenge persists, the cellular and fluid infiltration
continues to develop and inflammatory cells soon occupy a significant
volume of inflamed gingival tissues.
NEUTROPHILS

Release potent
Phagocytose lysosomal
Release large enzymes,
and kill bacteria
quantities of MMP’s cytokines, ROS
leading to breakdown causing further
of structural tissue
components of damage.
periodontium.

The opportunity for repair starts getting limited. The epithelium

continues to proliferate apically, deepening the pocket further (which
is rapidly colonised by subgingival bacteria.)
 As the epithelium proliferates , necrosis of epithelial
cells that are more distant from connective tissue
can lead to intraepithelial clefts and splits.

FIRST STAGE OF POCKET FORMATION

•A cycle of chronic inflammation sets in , characterized by

leucocytes, release of inflammatory mediators and destructive
enzymes, connective tissue breakdown and proliferation of
epithelium, in apical direction.
 As the epithelium proliferates , necrosis of epithelial
cells that are more distant from connective tissue
can lead to intraepithelial clefts and splits.

FIRST STAGE OF POCKET FORMATION

•A cycle of chronic inflammation sets in , characterized by

leucocytes, release of inflammatory mediators and destructive
enzymes, connective tissue breakdown and proliferation of
epithelium, in apical direction.
• It is an active process that results in a return to normal
haemostasis, and is mediated by specific molecules including a
class of endogenous lipid mediators, lipoxins, resolvins
and protectins.

i. Lipoxins: LX A4, B4. They also signal macrophages to

phagocytose the remnants of apoptotic cells at sites of
inflammation, without generating an inflammatory response.
ii. Resolvins: They inhibit neutrophilic infiltration
transmigration, they inhibit and production of
mediators. inflammatory
iii. Protectins: They reduce cytokine expression and also inhibit
neutrophilic infiltration.
 IMMUNE RESPONSES IN PERIODONTAL
PATHOGENESIS
Immunity is defined as the resistance of a host to pathogens and their
toxic products.

Innate factors such as complement, resident leukocytes and mast cells

play an important role in signaling endothelium, thus initiating
inflammation.

Acute inflammatory cells (i.e. neutrophils) protect local tissues by

controlling the periodontal microbiota within the gingival crevice and
junctional epithelium.

Chronic inflammatory cells, protect the entire host from within

the
subjacent connective tissues .
Schematic illustration of the changes in the gingival tissues during the
development of gingivitis and periodontitis
 Resistance to infection which an individual possesses
by virtue of his genetic and constitutional make up.
Prior contact with microorganisms or their products is
not essential.
Refers to the elements of the immune response that
are determined by inherited factors (innate).
Limited specificity
🞂 “Fixed” (they do not change or improve during an
immune response or as a result of previous exposure to the
pathogen.
They are activated when the primary defenses are
breached.
If innate responses fail to eliminate infection, then the effecter
cells adaptive immunity (lymphocytes) are activated.
Saliva secreted from three major salivary glands as well as from
numerous minor glands has an important role in maintaining oral health.
It contains molecular components that contribute to host defenses against
bacterial colonization and PDL disease.
These components include

That inhibit That

Molecules specific
that non inhibit
virulence back cell
specifically factors eg;
inhibit growth
Histatins and
plaque that
formation induce
neutralise cell death
LPS
Saliva contains IgA to PDL pathogens that target specific Ag and
inhibit bacterial adherence
 These are main initial site of interaction between plaque bacteria
and host.
 The keratinized epithelium of sulcular and gingival epithelial
provides protection for underlying PDL tissues ; also acts as barrier
against bacteria and their products.
 Epithelial cells stimulated with bacterial components and cytokines
produce
MMP’S

contribute to loss of connective tissue.

 Epithelial cells express antimicrobial peptides and the synthesis and
secretion of these molecules is up regulated in response to
periodontal bacteria.
These antimicrobial peptides have a wider role in regulating
innate and adaptive immune responses to infection.
These have chemokine-like activity, stimulating the
chemotaxis of a range of leukocytes involved in innate and
acquired immunity.
GINGIVAL CREVICULAR FLUID
It has a flushing action in gingival crevice but also likely
factions to bring the blood components of host defenses in the
sulcus
Flow of GCF increases in inflammation, and neutrophils are
an especially important element of GCF in health and disease.
Penetration of PDL tissue by plaque, bacteria and their products

Sentinel cells recognize their presence and signal protective immune

responses

Dendritic cells express a range of pattern recognition receptors(PRPs)

that interact with specific molecular structures on microbes –
MAMP’s

Activation of immune response to provide immediate

protection Excessive and inappropriate immune

 It is slower and reliant on complex interactions between
antigen presenting cells, T and B lymphocytes.
Key elements:
i. Antigen specificity of the responses that facilitate specific
targeting of a diverged range of effector elements (cytotoxic
T cells and antibodies)
ii. The ability to improve during exposure to antigen.
T cells are predominant in gingivitis and stable periodontal
lesions.

In contrast, in active periodontitis, B cells predominate and

are associated with pocket formation and progression of
disease.
MHC is a locus on short arm of chromosome 6 (6p21.3) that
encodes MHC classes I,II, III, which are involved in antigen
uptake, processing and presentation.

MHC class I molecules: present intracellular antigens

to CD8+ T cells and NK cells.
MHC class II molecules: present extracellular antigens
to CD4+ Tells.
MHC class III molecules include complement factors B, C2
and C4.
Includes B cells, macrophages, dermal dendritic cells
and Langerhan cells

These cells naturally express MHC-II molecules to activate

specific effector T cells.

Generate antigen specific immune response to

periodontal pathogens.
 Th1 Cells secrete
Th2 cells secrete

IFN gamma IL-

4,5,13.

Activates Cell Mediated Immunity

Activates Humoral (Antibody) mediated
Immunity.

Macrophages NK cells CD8+ cells

Specific antibodies are produced in response to increase in
bacterial challenge in periodontal disease.
End point of B cell activation.
High levels occur in GCF and these are produced locally by
plasma cells in periodontal tissues.
Antibodies to periodontal pathogens are primarily IgG
with few IgM or IgA types produced.
 CONCEPT OF HOST SUSCEPTIBILITY
Immune responses to the bacterial challenge never occur in isolation but take
place in the context of other host and environmental factors that influence
these responses and thereby determine the progression of disease.

HYPER-INFLAMMATORY RESPONSE TRAIT

The hyper-responder concept was originally proposed in the context of

responsiveness of monocytes to LPS challenge, suggesting the subjects
with disease possess an individual hyper-responsiveness monocytic trait,
characterized by elevated levels of inflammatory mediators released from
monocytes in response to bacterial challenge.
Individuals are considered normal for a given bacterial challenge in which
there is production of certain levels of inflammatory mediators in
periodontal tissues.
Those who are hyper-responders, the same bacterial challenge results in a
greater inflammatory response which over time would produce increased
tissue damage. & earlier presentation of clinical signs of disease.
 DOSE-RESPONSE CURVE

 A certain level of bacterial challenge results in a moderate release of

inflammatory mediators which together with infiltrating defense cells have
a protective role in eliminating bacteria in the sulcus and do not trigger
periodontal disease breakdown.

 Such a steady-state scenario may persist indefinitely.

 But any alteration in the quantity and quality of bio-film or alteration of

host defense leads to increased secretion of inflammatory mediators
leading to histopathologic changes.
 There is a threshold therefore between stable and active
disease, and this varies from person to person.
 The dose-response curve for any individual can shift to left or
right according to environmental changes.
 A shift to the left would result in increase in quantities of
inflammatory mediators produced for a given bacterial
challenge. And potentially an exacerbation of disease.
 A shift to right will have an opposite effect.
 In all cases, an increase in the LPS challenge would have the
tendency to the production of inflammatory
mediators, which
increase tip the site from a stable to a
progressing
may periodontal lesion.
 MODEL FOR PATHOGENESIS OF PERIODONTITIS
 Plaque bacteria initiate the inflammatory response but most of

the tissue damage results from the host response which is

influenced by genetic factors and environmental and acquired
risk factors.
 These factors, such as smoking, or genetic risk factors alter the

progression of the immune-inflammatory response and shift

the balance toward increased periodontal breakdown.
 This model implies that the presence of plaque bacteria does

not inevitably lead to tissue destruction and is supported by a

large number of epidemiologic studies, which confirm that
more advanced disease is usually confined to a minority of the
population.
 Biologic systems model

Represents periodontal pathogenesis, which involves

bacterial
components, environmental factors, specific
inflammatory
and host-genetic variations associated
mechanisms, with
disease.includes a person level, a genetic/epigenetic level, the
System
biologic phenotype, and ultimately the clinical phenotype
REFERENCES

1.Carranza’s Clinical Periodontology, 11th edition

2.Periodontol 200 31:167-180,2003
3. Periodontol 2000 31:167-180,2003
4. Orbans , oral histology and embryology, 12th
edition
5. Acta Odontol Scand 65-13, 2007